Analysis Device

ABSTRACT

An analysis device is disclosed which includes an electron detection medium to obtain information needed for analyzing an analyte in correlation with an electron transfer level, and a reagent part which is disposed on the electron detection medium and includes an electron transporting substance to transport electrons between the analyte and the electron detection medium, the electron transporting substance including a water-soluble aromatic heterocycle compound, and being free of a metal complex. An analysis method using the analysis device is also disclosed.

BACKGROUND

1. Technical Field

The present invention relates to an analysis device.

2. Related Art

As methods for measuring various substances contained in biologicalsamples such as blood, for example, methods with enzyme sensors usingenzymes are known. In an enzyme sensor utilizing an electrochemicalreaction system in which a general enzyme is used, a signal is obtainedby detecting, on an electrode surface, a transfer of electrons generatedbased on an enzymatic catalytic reaction. Even when a colorimetricreaction system, which depends on changes in the optical properties of apigment, is used as a detection method, its basic reaction is derivedfrom the enzymatic catalytic reaction (oxidation-reduction) and involveselectron transfer. In these reaction systems, electron transportefficiency in the reaction systems influences detection sensitivities.Various techniques in which the electron transport efficiency isimproved in order to increase the sensitivity of enzyme sensors areknown.

For example, National phase publication (Translation of PCT Application)No. 2002-514305 discloses a sensor in which a surface of an electrode ismodified with a substance having a helical structure, such as nucleicacid, as a conductive polymer, to promote electron transfer to or froman enzyme molecule to be targeted.

Since electron transfer between an electrode and an enzyme occursthrough the active center of the enzyme, the manner of the arrangementof a site, in which the active center is present, with respect to theelectrode is important. In order to decrease influences on theorientation of the active center of an enzyme and therefore to transportelectrons efficiently, molecules which function like an electrontransport mediator which is oxidized/reduced to transport electrons, forexample, complexes having, as an active center, a transition metal suchas iron, copper, osmium, or ruthenium, are known, and sensors using sucha complex are also known (for example, see Japanese National phasepublication (Translation of PCT Application) Nos. 2006-509837 and2005-520172).

As an enzyme electrode, a system in which a polypyrrole is used is known(for example, Biosensors & Bioelectronics, Vol. 7, (1992) pp. 461-471and Sensors and Actuators B, Vol. 106, (2005) pp. 289-295). Sincepyrrole, which is a monomer, has poor water-solubility, it is notpossible to directly mix pyrrole with an enzyme liquid to prepare anenzyme electrode on the surface of an electrode material. Therefore, forexample, in these documents (i.e., Biosensors & Bioelectronics, Vol. 7,(1992) pp. 461-471 and Sensors and Actuators B, Vol. 106, (2005) pp.289-295), a polymerization reaction is carried out using ferric chlorideand a pyrrole solution in a track etched membrane and, thereafter, themembrane is impregnated with an enzyme liquid to obtain an electrode.

SUMMARY OF THE INVENTION

However, there is still room for improvement with respect to thesensitivity of analysis devices such as sensors for detecting analytesin samples. In a sensor, a higher detection sensitivity not only allowsdetection of a small amount of analyte in a sample, but also becomesadvantageous when miniaturization of the sensor in itself is required.In addition, a metal complex used as an electron transport mediator isgenerally an expensive material and may become unstable as a substanceor interfere with a reaction potential since the metal complex in itselfis oxidized and reduced.

Further, with respect to an enzyme electrode in which a polypyrrole isused, a manufacturing method thereof is complicated. Moreover, whenvoltage is continuously applied to polypyrrole in an aqueous systemenvironment, polypyrrole may be decomposed (see Sensors and Actuators B,Vol. 106, (2005) pp. 289-295), and, therefore, the long-time usereliability may be low when using an enzyme electrode in whichpolypyrrole is used.

Thus, an analysis device that has higher sensitivity and reliabilitythan those conventionally used in the art by using an electrontransporting substance with stability has been demanded.

Accordingly, it is an object of the present invention to provide ananalysis device that has higher sensitivity and reliability than thoseconventionally used in the art; and also to provide an analysis methodusing the analysis device.

Exemplary embodiments of the present invention include the followings,but the present invention is not limited to the following exemplaryembodiments.

<1> An analysis device comprising:

an electron detection medium to obtain information needed for analyzingan analyte in correlation with an electron transfer level; and

a reagent part which is disposed on the electron detection medium andcomprises an electron transporting substance to transport electronsbetween the analyte and the electron detection medium, the electrontransporting substance comprising a water-soluble aromatic heterocyclecompound, and being free of a metal complex.

<2> The analysis device according to <1>, wherein the electrontransporting substance comprises at least one water-soluble aromaticheterocycle compound selected from the group consisting of pyridinecompounds and imidazole compounds (the group consisting of pyridine,imidazole and derivatives thereof).

<3> The analysis device according to <1> or <2>, wherein thewater-soluble aromatic heterocycle compound has a molecular weight of1,000 or less.

<4> The analysis device according to any one of <1> to <3>, wherein thereagent part further comprises an electron transfer compound thattransfers an electron to or from the analyte.

<5> The analysis device according to <4>, wherein the electron transfercompound is an oxidoreductase.

<6> The analysis device according to <4> or <5>, wherein a concentrationof the electron transporting substance is a concentration that allowstransportation of electrons between the electron detection medium andthe electron transfer compound.

<7> The analysis device according to any one of <1> to <6>, wherein aconcentration of the electron transporting substance is from 10 mass %to 60 mass % based on a total mass of the reagent part.

<8> The analysis device according to any one of <1> to <7>, wherein theelectron transporting substance comprises at least one water-solublearomatic heterocycle compound selected from the group consisting ofpyridine and aminomethyl pyridine.

<9> The analysis device according to any one of <1> to <8>, wherein thereagent part comprises a crosslinked substance.

<10> The analysis device according to any one of <1> to <9>, wherein thereagent part comprises a crosslinked substance obtained by crosslinkingusing at least one selected from the group consisting of glutaraldehyde,carbodiimide compounds and succinimide esters.

<11> The analysis device according to any one of <1> to <10>, whereinthe electron detection medium is an electric conductor.

<12> The analysis device according to any one of <1> to <11>, whereinthe analyte is a saccharide.

<13> An analysis method comprising obtaining information needed foranalyzing an analyte based on an electron transfer level of the analyteusing the analysis device according to any one of <1> to <12>.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the present invention are described in detailbased on the following figures, wherein:

FIG. 1 is a conception diagram of an analysis device according to anexemplary embodiment of the present invention;

FIG. 2 is a graph indicating glucose response currents from enzymeelectrodes in Example 1 of the present invention;

FIG. 3 is a voltammogram (oxidation wave) indicating glucose responsecurrents from enzyme electrodes in Example 2 of the present invention;

FIG. 4 is a graph indicating glucose response currents from enzymeelectrodes in Example 3 of the present invention;

FIG. 5 is a graph in which stability is confirmed in continuousmeasurement of enzyme electrodes in Example 4 of the present invention;

FIG. 6 is a graph indicating glucose response currents from enzymeelectrodes in Example 5 of the present invention; and

FIG. 7 is a graph indicating glucose response currents from enzymeelectrodes in Comparative Example 1 of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

An analysis device according to the present invention includes: anelectron detection medium to obtain information needed for analyzing ananalyte in correlation with an electron transfer level; and a reagentpart which is disposed on the electron detection medium and includes anelectron transporting substance to transport electrons between theanalyte and the electron detection medium, the electron transportingsubstance including a water-soluble aromatic heterocycle compound, andbeing free of a metal complex.

An analysis method according to the present invention includes obtaininginformation needed for analyzing an analyte based on an electrontransfer level of the analyte using the analysis device.

According to the present invention, electrons are efficientlytransported between the analyte and the electron detection medium sincethe electron transporting substance in the reagent part includes awater-soluble aromatic heterocycle compound (hereinafter, may also besimply referred to as an “aromatic heterocycle compound”). Since thearomatic heterocycle compound may simply function as a field fortransporting electrons and it is not necessary that the aromaticheterocycle compound in itself is subjected to oxidation-reduction as inthe case of a metal complex, the aromatic heterocycle compound isconsidered to have no dependency on a reaction potential in a system andto be a stable compound. As a result, the analysis device according tothe present invention including the reagent part containing such awater-soluble aromatic heterocycle compound can have higher sensitivityand reliability than those conventionally used in the art, for example,when being applied to a sensor and/or the like.

In addition, by using such an analysis device, an analyte can beanalyzed with better sensitivity and higher reliability than thoseconventionally used in the art.

More specifically, it is supposed that, when the water-soluble aromaticheterocycle compound is present in the reagent part, the molecules ofthe aromatic heterocycle compound are close to each other, the electroncloud of pi-electrons present to be perpendicular to the surfaces of themolecules of the aromatic heterocycle compound becomes anelectron-transporting path, and, therefore, electrons are efficientlytransported. In addition, it is supposed that the reason for notinvolving any oxidation-reduction reaction of the water-soluble aromaticheterocyclic compounds in itself is that the electron cloud ofdelocalized pi-electrons is utilized as the electron-transporting path.

An analysis device according to an exemplary embodiment of the presentinvention is described taking as an example the case of using as anenzyme electrode, with reference to FIG. 1. However, the presentinvention is not bound by the following theory.

An electron transfer layer 14 as the reagent part is disposed on anelectrode 12 as the electron detection medium in the enzyme electrode 10and molecules of electron transfer compound(s) such as a oxidoreductase16 are present to be independent of each other in the electron transferlayer 14. The active centers 18 of the molecules of the oxidoreductase16 are not oriented in the same direction with respect to the electrode12 because the active center 18 is often localized in a part of amolecule of the oxidoreductase 16. That is, in one molecule of theoxidoreductase, the active center 18 is placed in the vicinity of theelectrode 12, and in another molecule of the oxidoreductase 16, theactive center 18 is placed in a place away from the electrode 12.Therefore, the distances of electron transfer (directions of arrows inFIG. 1) between the active centers 18 of the oxidoreductase 16 and theelectrode 12 are considered to be varied. When a distance from theactive center 18 of a molecule of the oxidoreductase 16 to the electrode12 is long, electron transfer between the oxidoreductase 16 and theelectrode 12 by the oxidoreductase 16 alone becomes difficult.

According to the exemplary embodiment of the present invention, it issupposed that an aromatic heterocycle compound 20 (nitrogen-containingaromatic heterocycle compound in FIG. 1) is present between anoxidoreductase 16 and the electrode 12 in the electron transfer layer14. Therefore, it is supposed that even when a distance between theactive center 18 of an oxidoreductase 16 and the electrode 12 is long,if an aromatic heterocycle compound 20 is present therebetween, thearomatic heterocycle compound 20 enters into a gap between theoxidoreductase 16 and the electrode 12 and the aromatic heterocyclecompound 20 and the oxidoreductase 16 overlap one another to constructan electron transfer pathway with the pi-electrons of the aromaticheterocycle compound 20.

Construction of such an electron transfer pathway allows electrontransfer via the electron transfer pathway with an aromatic heterocyclecompound 20 not only between an oxidoreductase 16 present at a positionnear to the electrode 12 and the electrode 12 but also between anoxidoreductase 16 present at a position farther away from the electrode12 and the electrode 12.

As a result, electrons depending on the amount of an analyte which ispresent are transferred between the oxidoreductase 16 and the electrode12 and are converted into the amount of the analyte in a sample by adetection system which is not illustrated, so that the presence orabsence and amount of the analyte can be detected.

In the analysis device, the concentration of the electron transportingsubstance in the reagent part may be a concentration that allowstransportation of electrons between the electron transfer compound andthe electron detection medium and, for example, the concentration of theelectron transporting substance may be from 10 mass % to 60 mass %,preferably from 10 mass % to 50 mass %, based on the total mass of thereagent part, when the electron transfer compound is present. When theconcentration of the electron transporting substance is such aconcentration, the molecules of the aromatic heterocycle compoundincluded in the electron transporting substance around the analyte canbecome close to each other to construct the electron transfer pathwaywith a thickness enabling better electron transport. As a result, theanalysis device with better sensitivity can be made.

In the analysis device, the aromatic heterocycle compound may be, forexample, a nitrogen-containing aromatic heterocycle compound and, forexample, is preferably at least one selected from the group consistingof pyridine, imidazole, and derivatives thereof. The analysis devicewith higher sensitivity can be made with, as the aromatic heterocyclecompound, the nitrogen-containing aromatic heterocycle compound, forexample, at least one selected from the group consisting of pyridine,imidazole, and derivatives thereof.

In the analysis device, the reagent part preferably includes an electrontransfer compound which transfers electrons to or from an analyte andthe electron transfer compound is, for example, an oxidoreductase. Anaturally-occurring substance or a substance derived from a living bodycan easily be detected with higher sensitivity by incorporating theelectron transfer compound such as an oxidoreductase into the reagentpart. In the analysis device, the analyte may be, for example, asaccharide, and in this case, a saccharide can be detected with goodsensitivity.

The term “step” as used herein encompasses not only an individual stepbut also a step in which an expected effect of this step is attainedeven when the step cannot be clearly distinguished from other step(s).

A numerical value range indicated by using “from A to B” as used hereinrefers to a range including A and B as the minimum and maximum values,respectively.

In the present invention, when a plurality of substances correspondingto one component are present in a composition, the amount of thecomponent in the composition means the total amount of the plurality ofsubstances present in the composition unless otherwise specified.

The present invention is described below.

The analysis device according to an exemplary embodiment of the presentinvention includes: an electron detection medium for obtaininginformation needed for analyzing an analyte in correlation with anelectron transfer level; and a reagent part which is disposed on theelectron detection medium and includes an electron transportingsubstance for transporting electrons between the analyte and theelectron detection medium, wherein the electron transporting substanceincludes a water-soluble aromatic heterocycle compound, with the provisothat the electron transporting substance is free of a metal complex.

The electron detection medium is for obtaining information needed foranalyzing an analyte in correlation with an electron transfer level, andpreferably includes a conductive layer which can transfer electrons toor from the analyte and a supporting member for defining or ensuringphysical characteristics of the electron detection medium, for example,defining a shape or ensuring rigidity, which are disposed in this order.

The reagent part is disposed on the electron detection medium andincludes the electron transporting substance for transporting electronsbetween the analyte and the electron detection medium. In the reagentpart, the electron transporting substance may be included in a layerplaced on the electron detection medium. A layer that corresponds to thereagent part and includes the electron transporting substance isreferred to herein as “electron transfer layer.”

Electron Detection Medium

(1) Supporting Member

The material of the supporting member may be either a material havinginsulating properties or a material having conductivity. As thesupporting member having insulating properties, a commercially availableengineering plastic such as polyethylene terephthalate, polyimide,polystyrene, or Duracon (registered trademark from Polyplastics Co.,Ltd.) may be used.

As the supporting member having conductivity, for example, conductivecarbon paper, a carbon fiber web, or a metal in plate, bar, or thin filmform (e.g., gold or platinum) may be used. When the supporting memberhaving conductivity is used, the supporting member may also function asa lead for taking an output from the analysis device.

The supporting member may be a member having sufficient rigidity inmeasurement. The supporting member may optionally have flexibility. Theform of the supporting member is not particularly limited. Thesupporting member may have, for example, the form of a film or a rod,the form may variously be changed depending on a purpose.

The thickness of the supporting member may generally be from 0.1 mm to 1mm, depending on the application of the analysis device, but is notlimited thereto.

(2) Conductive Layer

The conductive layer in the electron detection medium is placed on thesupporting member and includes a conductive substance which can transferelectrons to or from the electron transfer layer described below. Bycontaining such a conductive substance, there are such advantages that,for example, the electron detection medium becomes an electric conductorand electrons can easily be detected as electric signals.

As the conductive substance, which is not particularly limited, a knownsubstance which can transfer electrons may be used. Examples of suchsubstances may include carbon materials, metals, metal-supported carbon,and the like, and one substance may be used singly or two or moresubstances may be used in combination.

A carbon material used as the conductive material may be used in theform of carbon particles or a structure in which carbon particles arehigh-density arranged or integrated. Examples of such carbon particlesmay include activated carbon, graphite, carbon black, and particlesforming a higher-order structure, represented by diamond-like carbon, acarbon nanotube, or fullerene. Such structures in which carbon particlesare high-density arranged or integrated include glassy carbon, pyrolyticgraphite carbon, plastic-formed carbon, and/or the like. For example,such an advantage that molding to a desirable shape is enabled can beobtained by using such a carbon material. The carbon particles, whichhave primary particle sizes, for example, ranging from 3 nm to 150 nm,more preferably from 3 nm to 50 nm, are used. The conductive substancein which the carbon particles have such particle diameters has such anadvantage that the specific surface area of the electron detectionmedium is increased or three-dimensional interaction of a fine structuremay easily occur in electron transfer.

The metal as the conductive material may be present as metal particlesin the conductive layer. When the metal particles are used, the metalparticles may be present independently of the carbon particles orsupported on the carbon particles.

Typical examples of such metal may include noble metals such as platinum(Pt), rhodium (Rh), gold (Au), silver (Ag), palladium (Pd), ruthenium(Ru), iridium (Ir), or osmium (Os), and one of these noble metals may beused alone or two or more thereof may be used in combination.Preferably, platinum is used alone or platinum and any one or more othernoble metal(s) are used in combination.

When the metal is supported as the metal particles on the carbonparticles, the particle size of the metal such a size that allows themetal particles to be suitably supported on the carbon particles, forexample, a size of a colloid level ranging from 1 nm to 20 μm,preferably from 1 nm to 4 nm. The amount of the metal particlessupported on the carbon particles may be, for example, from 0.1 part bymass to 60 parts by mass based on 100 parts by mass of the carbonparticles. The amount of the metal particles supported on the carbonparticles of not less than 0.1 part by mass can further improvesensitivity while the amount of not more than 60 parts by mass of themetal particles may provide, for example, a better relationship betweenthe amount of the metal used and the sensitivity, and thus provide aneconomical advantage. Preferably, the amount of the metal particlessupported on the carbon particles is from 0.5 part by mass to 40 partsby mass based on 100 parts by mass of the carbon particles.

The metal contained in the conductive layer may be present as acomponent in a different layer from that of the carbon particles. Theconductive layer in this case may be composed of the layer having thecarbon particles (carbon-containing layer) and the layer having themetallic element (metal-containing layer). As the kinds of metals whichmay be contained in the metal-containing layer, the above-mentionedmetals may be applied as is. Such a metal-containing layer is preferablyplaced between the supporting member and the carbon-containing layer.

The forms of the above-mentioned carbon material and metal are notparticularly limited and may be the forms of particles and any of otherforms such as plate, rod, and thin film forms. The forms of the carbonand the metal may be identical to each other or different.

The thickness of the conductive layer may generally be from 0.01 μm to10 μm but is not limited thereto, although varying according to theapplication of the analysis device.

Electron Transfer Layer

The electron transfer layer includes the electron transporting substanceand preferably further includes an electron transfer compound whichtransfers electrons to or from an analyte.

The electron transfer layer may preferably be placed at a positionallowing electron transfer to or from the electron detection medium. Theelectron transfer layer may be disposed in contact with the electrondetection medium, but is not necessarily in contact with the electrondetection medium, and, for example, another layer which does notinterfere with electron transporting may be disposed between theelectron transfer layer and the electron detection medium.

The thickness of the electron transfer layer may generally be from 0.1μm to 5 μm but is not limited thereto, although varying according to theapplication of the analysis device.

(1) Electron Transfer Compound

The electron transfer compound in the analysis device may be a compoundwhich transfers electrons, depending on the amount of an analyte whichis present, to or from the analyte. Examples of such electron transfercompounds may include a compound involved in an enzyme reaction with ananalyte. These electron transfer compounds may appropriately be selecteddepending on the application of the analysis device.

The electron transfer compound is preferably an enzyme, furtherpreferably an oxidoreductase. In the analysis device using the enzyme asthe electron transfer compound, electrons transferred between an analyteand the electron transfer compound can simply and easily be assessed aselectrons depending on the amount of the analyte which is present, basedon an enzyme-substrate relationship. Therefore, the analysis device issuitable for quantitatively measuring the concentration of a certainanalyte in a sample, in which various substances are mixed, by aspecific enzymatic reaction.

The oxidoreductase is an enzyme that catalyzes an oxidation-reductionreaction and a single enzyme or a combination of a plurality ofdifferent enzymes may be used in the same analysis device depending onthe kind of an analyte and the details of detection of interest.Examples of the oxidoreductase include glucose oxidase (GOD), galactoseoxidase, bilirubin oxidase, pyruvate oxidase, D- or L-amino acidoxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthineoxidase, sarcosine oxidase, L-lactate oxidase, ascorbate oxidase,cytochrome oxidase, alcohol dehydrogenase, glutamate dehydrogenase,cholesterol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase(GDH), fructose dehydrogenase, sorbitol dehydrogenase, lactatedehydrogenase, malate dehydrogenase, glycerol dehydrogenase,17B-hydroxysteroid dehydrogenase, estradiol 17B-dehydrogenase, aminoacid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase,3-hydroxysteroid dehydrogenase, diaphorase, cytochrome oxidoreductase,catalase, peroxidase, and glutathione reductase.

Especially, an oxidoreductase for a saccharide is preferable, andexamples such oxideredutase include glucose oxidase (GOD), galactoseoxidase, glucose dehydrogenase (GDH), fructose dehydrogenase, andsorbitol dehydrogenase.

The amount of the enzymes used is not particularly limited and mayappropriately be set.

(2) Aromatic Heterocycle Compound as Electron Transporting Substance

The aromatic heterocycle compound included in the electron transferlayer is a water-soluble aromatic heterocycle compound. Incorporatingthe water-soluble aromatic heterocycle compounds in the electrontransfer layer allows transportation of electrons between the electrontransfer compound and the electron detection medium. An analysis devicewith favorable stability under aqueous environment, higher reliabilityand better sensitivity can be provided by incorporating such an aromaticheterocycle compound rather than a metal complex into the electrontransfer layer. In addition, there is also such an advantage that thearomatic heterocycle compound does not depend on a reaction potential ina system.

In the present invention, the term “water-soluble” means dissolving inpure water of 20° C. in a mass ratio of 6% or higher under theenvironment at 20° C.

Since the water-soluble aromatic heterocycle compound includes anaromatic heterocycle, the electron clouds of pi-electrons can be formedto transport electrons. The aromatic heterocycle may be a five- orsix-membered ring. The aromatic heterocycle may be a condensed ornon-condensed aromatic heterocycle. Examples of the hetero atom includea nitrogen atom, an oxygen atom, and a sulfur atom.

The aromatic heterocycle compound may have at least one substituent onthe aromatic heterocycle as long as the water-solubility of the compoundis not impaired.

Examples of the substituent on the aromatic heterocycle of the aromaticheterocycle compound may include halogen atoms, amino group, alkylgroups, alkenyl groups, and alkoxy groups.

These substituents may further have at least one substituent, examplesof which include ones mentioned as the examples of the substituents onthe aromatic heterocycle.

The aromatic heterocycle compound may be, from the viewpoint of, forexample, sensitivity, preferably a nitrogen-containing aromaticheterocycle compound, and examples of the nitrogen-containing aromaticheterocycle compound may include imidazole, pyrazole, pyridine,pyrimidine, purine, and derivatives thereof. The aromatic heterocyclecompound is preferably, from the viewpoint of, for example, sensitivity,pyridine, imidazole or a derivative thereof, and is more preferablypyridine or a derivative thereof. Examples of pyridine or a derivativethereof may include pyridine and aminomethyl pyridine. One of sucharomatic heterocycle compounds may be used singly, or two or morethereof may be used in combination. As the aromatic heterocyclecompound, especially, aminomethyl pyridine is further preferred from aviewpoint of, for example, the sensitivity of the analysis device.

The aromatic heterocycle compound is preferably an aromatic heterocyclecompound having a molecular weight of 1,000 or less, more preferably anaromatic heterocycle compound having a molecular weight of 800 or less.Therefore, in an embodiment, it is preferable that the aromaticheterocycle compound according to the present invention does notencompass an aromatic heterocycle compound having a molecular weight ofmore than 1,000, such as a polymer. When the aromatic heterocyclecompound having a molecular weight of 1,000 or less is used, bettertransportation of electrons between the electron transfer compound andthe electron detection medium may be attained.

The electron transfer layer may be a layer including the aromaticheterocycle compound(s) in a concentration that allows transportation ofelectrons between the electron transfer compound and the electrondetection medium. As used herein, “concentration that allowstransportation of electrons between an electron transfer compound and anelectron detection medium” means a concentration at which at leastbetween the electron transfer compound and the electron detection mediumin the electron transfer layer the electron cloud of pi-electrons ispresent to be delocalized. Thus, when only a part of the electrontransfer layer is in contact with the electron detection medium in theanalysis device, the concentration of the aromatic heterocyclecompound(s) may be a concentration that allows the transportation ofelectrons between the electron transfer compound and the electrondetection medium in the region of the electron transfer layer in contactwith the electron detection medium, and is not necessarily aconcentration in the whole electron transfer layer.

The concentration that allows transportation of electrons between theelectron transfer compound and the electron detection medium canspecifically be varied according to the kind or concentration of theelectron transfer compound used and, in an embodiment, the concentrationof the aromatic heterocycle compound(s) may be 10 mass % or more, from10 mass % to 60 mass %, preferably from 15 mass % to 50 mass %, based onthe total mass of the electron transfer layer (reagent part) (when thewhole electron transfer layer is in contact with the electron detectionmedium). The concentration of the aromatic heterocycle compound(s)according to the present invention means mass % based on the total massthat is the mass of the electron transfer layer including the aromaticheterocycle compound(s) together with an electron transfer compound andanother additive that has been developed on the electron detectionmedium and has been dried.

The aromatic heterocycle compound may be subjected to crosslinkingtreatment or contained in and coated with a polymer. By being subjectedto such treatment, the aromatic heterocycle compound is crosslinkedbetween the molecules thereof or the aromatic heterocycle compound iscrosslinked with an electron transfer compound, and can be maintained inan electron transfer layer for a long term. An analysis device havingthe electron transfer layer (reagent part) which is such a crosslinkedsubstance allows the aromatic heterocycle compound to remain around aconductive layer and an analyte, for example, even in continuousmeasurement for a long term, so that a relative position to theconductive layer or the analyte can be further favorably maintained, forexample, such an advantage as to be able to maintain a stable output isobtained.

The crosslinking treatment may be a crosslinking treatment that iscommonly used in crosslinking of, for example, protein. Examples ofcrosslinking agents used for such crosslinking treatment may includeglutaraldehyde and carbodiimide compounds, and succinimide esters. Oneof these crosslinking agents may be used alone or two or more thereofmay be used in combination.

The amount of such an added crosslinking agent is not particularlylimited if being a commonly used amount and may appropriately be set ina sufficient range such as, for example, 10 times or more that of amaterial to be crosslinked.

An electron transporting substance in the electron transfer layerincludes the aromatic heterocycle compound, the electron transportingsubstance is free of a metal complex. Example of such a metal complexwhich is not included in the electron transporting substance accordingto the present invention include metal complexes conventionally used asso-called electron transport mediators. Specific examples of such ametal complex include osmium complexes.

Other Layers

The analysis device according to the present invention may includeanother layer(s) at any position as far as electron transfer between theelectron detection medium and the electron transfer layer is possible.Examples of such other layers may include a protective layer, a layerfor restricting penetration of substances, and a functional layer formodifying the surface of the electron detection medium; and the analysisdevice may include one or two or more in combination thereof.

The protective layer is not particularly limited as long as being ableto protect the surface of the electron transfer layer or the analysisdevice. Examples of the protective layer may include cellulose acetatepolymer, polyurea, polyamide, polyester, polyethylene oxide, polyvinylalcohol, and lipid bilayer membranes.

The thickness of the protective layer is not particularly limited andmay be, for example, from 0.5 μm to 5 μm.

Examples of a membrane for restricting penetration of substances in theanalysis device, which membranes are intended to adjust the detectionconcentration range of an analyte, may include polyurea, polyamide,polyester, polyethylene oxide, and polyvinyl alcohol.

The thickness of the membrane for restricting penetration of substancesis not particularly limited and may be, for example, from 0.5 μm to 5μm.

Examples of the functional layers for modifying the surface of theelectron detection medium, which layers are intended to improve thereactivity of a conductive member, may include thiol compounds, silanecoupling agents, and lipid bilayer membranes.

The thickness of the functional layer is not particularly limited andmay be, for example, from 0.001 μm to 5 μm.

Analyte

An analyte to be detected by the analysis device is not particularlylimited as long as being a substance which can transfer electrons,depending on the amount of an electron transfer compound which ispresent, to or from the electron transfer compound and may beappropriately set according to the kind of the electron transfercompound.

For example, when the analysis device is used in a clinical application,various substrates contained in a clinical sample may be analytes.Examples of such clinical sample may include blood, serum, plasma,interstitial fluid, urine, sweat, tears, and saliva. Typical examples ofthe substrate may include glucose, uric acid, and glycosylated proteins.

For example, when the analysis device is utilized in a non-clinicalapplication such as monitoring of fermentation, control of an industrialprocess, or environmental monitoring (e.g., suppression of efflux ofliquid and gas and contamination), a food test, or veterinary medicine,various substrates contained in a non-clinical sample such asfermentation liquid, effluent, waste fluid, food, or milk may be ananalyte.

Method for Producing Analysis Device

As a method for producing an analysis device, which is not particularlylimited, any method in which each layer or each member as describedabove can be placed may be applied.

For example, by molding, shaping, or printing a mixture of componentsfor a conductive layer, the conductive layer may be disposed on asupporting member to form an electrode, followed by disposing a layer ofa liquid reaction mixture for an electron transfer layer containing anenzyme and an aromatic heterocycle compound.

In a case where crosslinking treatment of an electron transfer compoundand an aromatic heterocycle compound in an electron transfer layer isperformed, the method of crosslinking may vary depending on the kinds ofthe crosslinking agent and the aromatic compound. In an embodiment, acrosslinking agent may be incorporated into a reaction mixture, and thecrosslinking may be performed simultaneously with forming an electrontransfer layer. Alternatively, the crosslinking agent may beincorporated into a treatment liquid which is different from thereaction mixture, and after an analysis device is obtained withoutcrosslinking as described above, the treatment liquid containing thecrosslinking agent is applied to the electron transfer layer by adding,spraying, or immersing the liquid to perform crosslinking treatment.

As a print medium, for example, a film- or plate-like print medium maybe used. The analysis device may be used after being removed from theprint medium or may be used while being supported on the print medium.In the latter case, the print medium may function as a supportingmember. The print medium may include a recess which is formed in theprinted part of the mixture. In this case, a mask may be omitted.

It will be appreciated that drying during the production is preferablyperformed at a lower temperature than a temperature at which substantialdeactivation of an enzyme occurs.

The analysis device according to the present invention can basically beformed only in a simple step such as forming a mixture, molding, anddrying. That is, it can be expected to reduce a production cost to sucha degree that a high mass-production technology can be utilized and adisposable analysis device can be produced.

The analysis method according to the present invention includesobtaining information needed for analyzing an analyte based on theelectron transfer level of the analyte using the analysis device(referred to as an information obtaining step). In the analysis method,information for analyzing the analyte can be obtained with goodsensitivity and high reliability because the analysis device asdescribed above is used.

In the information obtaining step in the analysis method, theinformation needed for performing analysis is obtained based on theelectron transfer level of the analyte. As used herein, “informationneeded” includes, for example, the amount, kind, and oxidation-reductionstate of the analyte, time-dependent change in the analyte, and thelike.

The analysis device as described above as an exemplary embodimentincludes the conductive layer in the electron detection medium but theelectron detection medium in the analysis toll of the present inventionis not limited thereto. For example, the electron detection medium mayinclude a color former.

When the electron detection medium includes a color former, the electrondetection medium preferably has a configuration in which the colorformer is retained in a porous material insoluble in a sample. Typicalexamples of such a porous material may include a gelled material of, forexample, polyacrylamide or polyvinyl alcohol. Examples of a color formerinclude MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide), INT(2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride),WST-4(2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt), and 4AA (4-aminoantipyrine).

Application

The analysis device according to the present invention is applicable tovarious applications because of having such an advantage that electronscan efficiently be transported to an electron detection medium throughan electron transporting substance. Examples of such applications mayinclude uses in, for example, an enzyme electrode; a sensor by which, asa platform, measurement of a substance by a colorimetric reaction systemutilizing a color former and/or the like is performed; or the reactionsystem of a bio-fuel cell.

EXAMPLES

The present invention is described in detail below with reference toExamples. However, the present invention is not limited thereto at all.Unless otherwise specified, “%” is based on a mass.

Example 1 (1) Production of Enzyme Electrode

For an enzyme electrode, Pt (Au) was sputtered on a polyimide (PI) filmto obtain a substrate having a platinum layer. As an electrode material,a printing ink in which 40 wt % Ketjen black (manufactured by LionCorporation) was mixed with 40 wt % polyester resin as a binder and 20wt % isophorone as a solvent was used. The surface of the polyimide filmwas printed with the printing ink so that the printing ink had athickness of 10 μm.

Then, an enzyme liquid containing 1,250 U/ml of a wild-type GDH solution(0.1M MES buffer solution), 1% adonitol as a stabilizer, and 1 wt % of4-aminomethyl pyridine as a water-soluble aromatic heterocycle compoundwas prepared.

The prepared enzyme liquid was dropwise added to the surface of theelectrode using a precise syringe, and thereafter, the resultingelectrode was left to stand for 4 hours under the environment of 23° C.and a relative humidity of <8% to be dried, whereby an enzyme electrodewas obtained.

As a comparative enzyme electrode, a comparative enzyme electrode wasproduced in substantially the same manner as described above except that4-aminomethyl pyridine was not added.

(2) Measurement by Enzyme Electrode

Electrode responses to glucose (100 mg/dL or 600 mg/dL) in 0.1 Mphosphate buffer (pH 7.4) at 23° C. and +0.6 V (vs. Ag/AgCl) weredetected by an amperometric method using the enzyme electrodes obtainedas described above. The results are shown in FIG. 2. In FIG. 2, theblack circles and the black triangles indicate measurements using theenzyme electrode added with 17% 4-aminomethyl pyridine and thecomparative enzyme electrode added with no 4-aminomethyl pyridine,respectively.

As indicated in FIG. 2, about 13 times higher current density wasdetected in the glucose response current from the enzyme electrodecontaining 4-aminomethyl pyridine than that in the response current fromthe comparative enzyme electrode added with no 4-aminomethyl pyridine.This indicates that sensitivity is increased by using 4-aminomethylpyridine as the aromatic heterocycle compound.

Example 2 (1) Production of Enzyme Electrode

For an enzyme electrode, Au was sputtered on a polyetherimide (PEI) filmto obtain a substrate having a gold layer. As an electrode material, aprinting ink in which 40 wt % Ketjen black (manufactured by LionCorporation) was mixed with 40 wt % polyester resin as a binder and 20wt % isophorone as a solvent was used. The surface of the polyetherimidefilm was printed with the printing ink so that the printing ink had athickness of 10 μm, whereby a a working electrode was obtained.

Then, enzyme liquid containing 2,500 U/ml of a wild-type GDH solution(0.1 M MES buffer solution), 2% sucrose as a stabilizer, 1 v/v %glutaraldehyde as a crosslinking agent, and 1% of 4-aminomethyl pyridineas the aromatic heterocycle compound was prepared.

The prepared enzyme liquid was dropwise added to an electrode surfaceusing a precise syringe and the resulting electrode was left to standfor 10 minutes at a normal temperature (23° C.) and a normal relativehumidity (40% RH) to dry the surface. Then, the electrode washeat-treated at 40° C. for 15 minutes for drying, and then was left tostand for 2 hours under the environment of 23° C. and <2% RH for furtherdrying, whereby an enzyme electrode was obtained.

As a comparative enzyme electrode, a comparative enzyme electrode wasproduced in substantially the same manner as described above except that4-aminomethyl pyridine was not added.

(2) Measurement by Enzyme Electrode

Electrode responses to glucose in 0.1 M phosphate buffer (pH 7.4) weredetected by voltammetry using the enzyme electrode obtained as describedabove. The voltammetry was performed at a sweep speed of 20 mV/s usingthe enzyme electrode produced in the above described (1), Pt, andAg/AgCl as working, counter, and reference electrodes, respectively, ata measurement temperature of 37° C. A glucose concentration was 100mg/dL or no glucose was added.

The results are shown in FIG. 3 (third scan is indicated). In FIG. 3,the lozenges, the quadrangles, the circles, and the triangles indicatethe measurement results in the case of 100 mg/dL glucose using theenzyme electrode added with 4-aminomethyl pyridine, the measurementresults in the case of 0 mg/dL glucose using the enzyme electrode addedwith 4-aminomethyl pyridine, the measurement results in the case of 100mg/dL glucose using the comparative enzyme electrode added with no4-aminomethyl pyridine, and the measurement results in the case of 0mg/dL glucose using the comparative enzyme electrode added with no4-aminomethyl pyridine, respectively.

As indicated in FIG. 3, since a potential at which oxidization ofglucose begins to occur is around −0.2 V with or without the addition of4-aminomethyl pyridine and any specific peaks are not observed in theother potential regions, the oxidation-reduction of 4-aminomethylpyridine in itself is not considered to occur.

Thus, it was exhibited that, in the enzyme electrodes added with4-aminomethyl pyridine, oxidation-reduction of a compound in itself didnot occur to transport electrons as in the case of a metal complex, andglucose was able to be stably detected with good sensitivity in theabsence of an electron transfer mediator such as a metal complex.

Example 3

Enzyme electrodes 3A to 3F were obtained in substantially the samemanner as in Example 2 (1) except that the concentrations of4-aminomethyl pyridine were in the range of from 1% to 6% in preparationof enzyme solutions. In the final form of the enzyme electrodes, theconcentrations of from 1% to 6% correspond to dry masses of from 23% to64%, respectively.

Measurement for 100 mg/dL glucose in 0.1 M phosphate buffer (pH 7.4) wasperformed using the enzyme electrodes 3A to 3F and the comparativeenzyme electrode containing no 4-aminomethyl pyridine produced inExample 2 (1), at a measurement temperature of 37° C. The enzymeelectrodes 3A to 3F and the comparative enzyme electrode; Pt; andAg/AgCl were used as working, counter, and reference electrodes,respectively. The results are shown in FIG. 4.

As indicated in FIG. 4, it is found that glucose can be detected bysetting the concentrations of 4-aminomethyl pyridine added in the enzymesolutions to the range of from 1% to 6% (from 23% to 64% on a dry massbasis in final form). It is also found that respective sensitivities areimproved by setting the concentrations of 4-aminomethyl pyridine intothe range of from 2% to 5% (from 38 to 60% on a dry mass basis in finalform), especially to 4% (55% on a dry mass basis in final form).

Example 4

Crosslinked enzyme electrodes 4A and 4B were obtained in substantiallythe same manner as in Example 2 (1) except that after dropwise adding ofthe prepared enzyme liquid to the electrode surface using a precisesyringe and drying, glutaraldehyde was added to perform crosslinkingtreatment.

The crosslinking treatment was performed as described below. A driedelectrode surface was gently rinsed with dH₂O and the electrode wasimmersed in 1 v/v % glutaraldehyde (GA) solution (containing 1%adonitol). After the immersion for 45 minutes or 120 minutes, eachelectrode was taken out and incubation was performed overnight in theenvironment of a room temperature and a low humidity (23° C. and <2%RH). As a result, each of the enzyme electrodes 4A (treatment time of 45minutes) and 4B (treatment time of 120 minutes) was obtained.

Continuous measurement for glucose at a predetermined concentration wasperformed using each of the obtained enzyme electrodes 4A and 4B. Forthe continuous measurement, electrode responses to glucose in aphosphate buffer were detected by an amperometric method at 25° C. and+0.6 V (vs. Ag/AgCl) for 100 mg/dL glucose. The evaluation for a changein relative value over time, based on a current density just after thestart of the measurement as an initial value of 100%, was performed. Theresults are shown in FIG. 5. In FIG. 5, the quadrangles, the triangles,and the lozenges indicate the cases of using the enzyme electrode 4A,the enzyme electrode 4B, and an uncrosslinked enzyme electrode,respectively.

As indicated in FIG. 5, it is found that, by performing the crosslinkingtreatment of 4-aminomethyl pyridine with glutaraldehyde, thelocalization of 4-aminomethyl pyridine is suppressed, the efflux fromthe electron transfer layer can also be prevented, and more stableoutputs than that from the untreated enzyme electrode is maintained.

Example 5

Electrode responses to glucose were detected in substantially the samemanner as in Example 1 except that 5-amino-4-imidazolecarboxamide/HCl,instead of 4-aminomethyl pyridine, was added to be 17%. The results areshown in FIG. 6. In FIG. 6, the black quadrangles and the black lozengesindicate the cases of using the enzyme electrode added with 17% of5-amino-4-imidazolecarboxamide and the comparative enzyme electrodeadded with no aromatic heterocycle compound, respectively.

As indicated in FIG. 6, it is found that the current densities arefurther favorably detected compared with the comparative enzymeelectrode by using 5-amino-4-imidazolecarboxamide/HCl instead of4-aminomethyl pyridine, as in the case of using 4-aminomethyl pyridine,and it was found that glucose can be detected with good sensitivity byusing an imidazole derivative.

Comparative Example 1

Enzyme electrodes in Comparative Example were obtained in substantiallythe same manner as in Example 4 except that from 0.01% to 1.0% ofpoly(2-vinylpyridine) (weight molecular weight of about 21,000, FlukaCorporation) was used instead of 4-aminomethyl pyridine. Responses to100 mg/dL of glucose were compared with relative values for an enzymeelectrode containing no water-soluble aromatic heterocycle compound insubstantially the same manner as in Example 4 except that thecomparative enzyme electrodes were used. The results are shown in FIG.7.

As indicated in FIG. 7, it was found that the responsiveness in the caseof using poly(2-vinylpyridine) was similar to that in the enzymeelectrode containing no water-soluble aromatic heterocycle compound or,at a concentration of 0.1% or more, was lower than that in the case ofusing the enzyme electrode containing no water-soluble aromaticheterocycle compound.

As described above, it is found that glucose in a sample can be detectedwith high sensitivity without treatment of adjusting the orientation ofenzymes and without using any metal complex and/or the like, by usingthe enzyme electrodes of the present examples containing 4-aminomethylpyridine. In addition, it is found that stable measurement is possiblesince 4-aminomethyl pyridine in itself is not involved inoxidation-reduction. Furthermore, the enzyme electrodes are hard to beaffected by a reaction potential, unlike a metal complex and/or thelike, and are applicable to a wide application such as a sensing devicein a clinical situation represented by SMBG (self-monitoring of bloodglucose), continuous blood glucose monitoring (CGM), or the like, areaction system such as a bio-fuel cell, or a sensing device forindustrial or environmental use in a non-clinical situation.

All references, patent applications, and technical standards describedin the present specification are herein incorporated in their entiretyby reference into the specification, to the same extent as if eachindividual reference, patent application or technical standard wasspecifically and individually indicated to be incorporated herein byreference.

1. An analysis device comprising: an electron detection medium to obtaininformation needed for analyzing an analyte in correlation with anelectron transfer level; and a reagent part which is disposed on theelectron detection medium and comprises an oxidoreductase that transfersan electron to or from the analyte, and an electron transportingsubstance to transport electrons between the oxidoreductase and theelectron detection medium without involving any oxidation-reductionreaction, wherein the electron transporting substance is a water-solublearomatic heterocycle compound that is free of a metal complex.
 2. Theanalysis device according to claim 1, wherein the electron transportingsubstance comprises at least one water-soluble aromatic heterocyclecompound selected from the group consisting of pyridine compounds andimidazole compounds.
 3. The analysis device according to claim 1,wherein the water-soluble aromatic heterocycle compound has a molecularweight of 1,000 or less. 4-5. (canceled)
 6. The analysis deviceaccording to claim 1, wherein a concentration of the electrontransporting substance is a concentration that allows transportation ofelectrons between the electron detection medium and the oxidoreductase.7. The analysis device according to claim 1, wherein a concentration ofthe electron transporting substance is from 10 mass % to 60 mass % basedon a total mass of the reagent part.
 8. The analysis device according toclaim 1, wherein the electron transporting substance comprises at leastone water-soluble aromatic heterocycle compound selected from the groupconsisting of pyridine and aminomethyl pyridine.
 9. (canceled)
 10. Theanalysis device according to claim 1, wherein the reagent part comprisesa crosslinked substance obtained by crosslinking using at least oneselected from the group consisting of glutaraldehyde, carbodiimidecompounds and succinimide esters.
 11. The analysis device according toclaim 1, wherein the electron detection medium is an electric conductor.12. The analysis device according to claim 1, wherein the analyte is asaccharide.
 13. An analysis method comprising obtaining informationneeded for analyzing an analyte based on an electron transfer level ofthe analyte using the analysis device according to claim
 1. 14. Theanalysis device according to claim 1, wherein the electron transportingsubstance comprises an electron cloud of pi-electrons perpendicular tothe surfaces of the molecules of the electron transporting substance.15. The analysis device according to claim 1, wherein the electrontransporting substance comprises 4-aminomethyl pyridine or5-amino-4-imidazolecarboxamide.
 16. The analysis device according toclaim 1, wherein the electron transporting substance has a molecularweight of 800 or less.
 17. The analysis device according to claim 1,wherein the oxidoreductase and the electrode have a gap in between, andthe electron transporting substance is configured to enter into the gapin the presence of the analyte.
 18. The analysis device according toclaim 1, wherein the electron transporting substance is configured toenter into a gap between the oxidoreductase and the electrode, and theelectron transporting substance and the oxidoreductase overlap oneanother to construct an electron transfer pathway with pi-electrons ofthe electron transporting substance in the presence of the analyte. 19.The analysis device according to claim 1, wherein the electrontransporting substance is not a polymer.
 20. An analysis methodcomprising transporting electrons in a reagent part of an analysisdevice by electron transporting substances between an analyte and anelectron detection medium without involving oxidation-reduction of theelectron transporting substances, wherein the reagent part is disposedon the electron detection medium and comprises an oxidoreductase thattransfers an electron to or from the analyte, and the electrontransporting substance is a water-soluble aromatic heterocycle compoundthat is free of a metal complex; and obtaining information about theanalyte based on an electron transfer level of the analyte by thetransporting electrons step.